Application of tetrahydrocarbazole piperazine small molecular compound and salt thereof in preparation of antibacterial drugs

Abstract

The invention discloses an application of tetrahydrocarbazole piperazine small molecular compounds and salts thereof in preparation of antibacterial drugs, and belongs to the technical field of biological medicines. The compound provided by the invention can effectively inhibit staphylococcus aureus and drug-resistant staphylococcus aureus infection in vivo and in vitro. Meanwhile, the compounds have good sensibilization activity on antibacterial peptides, and have the unique advantage that the generation of drug resistance is not easily induced compared with existing antibiotics. The possible action target of the compound is a transporter Pmt responsible for secretion of bacterial toxin PSM, and no related inhibitor of the target is reported at present. The invention provides a brand-new thought and strategy for the research of novel anti-bacterial infection drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a class of tetrahydrocarbazole-piperazine small molecule compounds and their salts in the preparation of antibacterial drugs. Background Art

[0002] Bacterial drug resistance is a major public safety issue that seriously endangers human life and health. According to statistics, in 2021, bacterial drug resistance directly led to 4.21 million deaths globally. If not controlled, it is expected that drug-resistant infections will cause 10 million deaths annually by 2050. Among the six clinically common "superbugs" (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, collectively referred to as ESKAPE), Methicillin-Resistant Staphylococcus aureus (MRSA) is an important one, seriously threatening human life and health and ranking among the high-priority pathogens on the list of key bacterial pathogens released by the WHO in 2024. Research shows that the main drug resistance mechanisms of MRSA include hydrolase production, target site alteration, and active efflux. Among them, the efflux of antimicrobial peptides or antibiotics out of the cell by efflux pumps is an important mechanism for the formation of its drug resistance. Therefore, efflux pump inhibitors, as a strategy to enhance the sensitivity of antibiotics or delay the emergence of bacterial drug resistance, have become an important direction in the research and development of new antibacterial agents.

[0003] Research shows that there is an ATP-binding cassette transporter in MRSA, which not only acts as an efflux pump to transport natural antimicrobial peptides out of the cell, forming the resistance of Staphylococcus aureus to natural antimicrobial peptides and immune escape, but also is responsible for transporting the key virulence factor of MRSA - Phenol-Soluble Modulins (PSM) out of the cell to exert its multiple pathogenic effects such as cell lysis, pro-inflammation, hemolysis, and biofilm formation. Therefore, this transporter is also called Pmt (Polyol / Monosaccharide Transporter). Research also found that Pmt is highly conserved in the genus Staphylococcus. Given that Pmt combines two key immune escape mechanisms, the formation of MRSA antimicrobial peptide resistance and the secretion of the important toxin PSM, and its high conservation in the genus Staphylococcus, Pmt has become a highly promising target for the development of anti-MRSA drugs.

[0004] In 2020, the complete crystal structure of the Pmt protein was resolved, revealing the crystal structure of the key domain PmtCD for exporting antimicrobial peptides and transporting PSM and its complex with the ligand ATPγS, and elucidating its mechanism of action. Research has shown that PmtCD is mainly composed of a transmembrane domain (TMD) and a nucleotide-binding domain (NBD) coupled together. Its transport process mainly includes the following steps: ① In the resting state, the lipids filling between the two TMDs are separated, and the transport system is in a closed state; ② The PSMα-helical peptide produced by bacteria can directly enter the membrane or enter the lumen of the TMD through the cytoplasmic pathway; ③ ATP binds to the interface of the NBD dimer, bringing a pair of NBD domains closer while causing the homologous TMD to move inward, expelling PSM out of the cell; ④ ATP hydrolysis releases ADP, providing energy to change the protein conformation back to the resting state. Therefore, if a small molecule compound is designed to competitively bind to the cavity of the PmtCD protein, it can prevent the formation of the PmtCD-ATP complex, interrupt the energy supply for transport, thereby inhibiting the secretion of PSM toxins and also preventing the efflux of natural antimicrobial peptides by PmtCD. This will provide an important entry point and a unique research strategy for the research of new anti-MRSA drugs, but there has been no relevant report on PmtCD-targeted inhibitors so far. Summary of the Invention

[0005] The object of the present invention is to provide the use of a class of tetrahydrocarbazole-piperazine small molecule compounds and their salts in the preparation of antibacterial drugs. This class of small molecule compounds can inhibit the secretion of key bacterial toxins and the efflux of natural antimicrobial peptides by targeting Pmt, thereby exerting dual antibacterial activities.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] One object of the present invention is to provide the use of a class of tetrahydrocarbazole-piperazine small molecule compounds or their salts in the preparation of antibacterial drugs or antibacterial drug sensitizers. The tetrahydrocarbazole-piperazine small molecule compounds or their salts are represented by the following structural formula (I):

[0008]

[0009] Wherein, n is 0, 1, 2 or 3;

[0010] R1, R2, R3 and R4 are any one of hydrogen, halogen, lower haloalkane, lower alkane, lower cycloalkane, hydroxyl, lower hydroxyalkane, lower alkoxy, phenyl, substituted phenyl, amino, lower alkylamino, lower haloalkylamino, lower cycloalkylamino, lower alkynylamino, nitro, lower nitroalkane, cyano, lower cyanoalkane, amide group, lower cycloalkylamide group and lower amide group alkyl;

[0011] R5 is hydrogen or an organic ketone compound;

[0012] *The marked position is a chiral carbon atom.

[0013] 2. The application according to claim 1, wherein the organic ketone compound includes any one of 1-((1H-1,2,4-triazol-5-yl)thio)propan-2-one, 1-morpholinopropan-2-one, (1-adamantan-1-yl)ethan-1-one, 1-(benz[d]oxazol-2-ylthio)propan-2-one, 1-(4,6-dimethylpyrimidin-2-yl)thio)propan-2-one, 1-((5-amino-1,3,4-thiadiazol-2-yl)thio)propan-2-one, 1-(4-fluorophenyl)propan-2-one, 4-acetyl-N,N-dipropylbenzenesulfonamide, 1-(4-phenylpiperazin-1-yl)propan-2-one, 1-(4-nitrophenyl)ethan-1-one, 1-(4-methylpiperazin-1-yl)propan-2-one, 7-(methylsulfonyl)-3,4-dihydroquinolin-2(1H)-one, N-phenylethanethioamide, N-(4-butoxyphenyl)ethanethioamide, 5-(methylsulfonyl)benzo[d]thiazol-2(3H)-one, pent-1-yne, 1-(piperidin-1-yl)butan-2-ol, but-1-yne, 4-(4-fluorophenyl)butan-2-one, 1-(4-(2-methoxyphenyl)piperazin-1-yl)propan-2-one, 1-(4-(3-chlorophenyl)piperazin-1-yl)propan-2-one, acetophenone, 1-(2-phenoxyphenyl)ethan-1-one, 1-(phenylthio)propan-2-one, 1-(dimethylamino)propan-2-one, furan-3-yl(4-(2-hydroxybutyl)piperazin-1-yl)methanone, 4-(3,4-dimethoxyphenyl)-2-methyl-4,5-dihydrothiazole, 1-(piperidin-1-yl)propan-2-one, 1-(diethylamino)propan-2-one and 1-(3-isopropyl-1H-pyrazol-5-yl)ethan-1-one.

[0014] Preferably, the lower cycloalkane is a ring containing 3 to 7 carbons; the lower substituent means that the corresponding aliphatic hydrocarbon group is straight-chain or branched-chain, saturated, and contains 1 to 4 carbon atoms.

[0015] Most preferably, the compound is any one of the following compounds:

[0016]

[0017] The tetrahydrocarbazolopyrazine small molecule compound reacts with an acid to form a salt, and the acid includes hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, acetic acid, tartaric acid, salicylic acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid or succinic acid, etc.

[0018] Preferably, the drug is an anti-Gram-positive bacterium drug.

[0019] Further preferably, the Gram-positive bacterium is Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0020] Preferably, the tetrahydrocarbazolopyrazine small molecule compounds and their salts exert antibacterial effects by acting on the target PmtCD.

[0021] Preferably, the tetrahydrocarbazolopyrazine small molecule compounds and their salts exert antibacterial effects by inhibiting the natural antibacterial peptide efflux pump.

[0022] Preferably, the tetrahydrocarbazolopyrazine small molecule compounds and their salts exert dual antibacterial activities by inhibiting the secretion of key toxin PSM and the efflux of natural antibacterial peptides.

[0023] The present invention may also consider providing an antibacterial pharmaceutical composition, which contains the above-mentioned tetrahydrocarbazolopyrazine small molecule compounds and their salts, and a pharmaceutically acceptable carrier. The pharmaceutical composition is configured as an injection, capsule, syrup, aerosol, gel, cream, pill or transdermal patch.

[0024] Preferably, the antibacterial pharmaceutical composition can be used alone or in combination with other drugs.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention discloses for the first time the application of small molecule compounds with a tetrahydrocarbazolopyrazine four-ring skeleton as the basic parent nucleus in the preparation of antibacterial drugs and antibacterial drug sensitizers. In particular, the compounds have good anti-Staphylococcus aureus activity in vitro and in vivo, especially against drug-resistant Staphylococcus aureus, such as MRSA, and also have good inhibitory effects.

[0027] The present invention also discloses the possible action targets of the above compounds, that is, by targeting Pmt, it inhibits the secretion of the bacterial key toxin PSM and the efflux of natural antibacterial peptides, thereby exerting dual antibacterial activities. The advantage of this target is that it is a Staphylococcus aureus virulence-related target and is not essential for bacterial growth. Therefore, compared with traditional antibiotics, it has the unique advantage of being less likely to induce drug resistance, and currently there are no reports on the research of inhibitors related to this target. Description of the Drawings

[0028] Figure 1 Growth curves of Staphylococcus aureus strains under the action of different concentrations of compound DY-210; among them, A is 29213; B is LAC; C is Mu50;

[0029] Figure 2Under the action of DY-210, the morphological changes of Staphylococcus aureus under the electron microscope; among them, A shows the morphological changes of bacteria under the scanning electron microscope under the action of different concentrations of DY-210; B shows the results of the morphological changes of bacteria under the transmission electron microscope under the action of different concentrations of DY-210.

[0030] Figure 3 Results of the protective effect of DY-210 on mice with sepsis caused by LAC; among them, A shows the effect of DY-210 on the survival rate of septic mice; B shows the effect of DY-210 on the histopathology of the organs of infected mice.

[0031] Figure 4 Graph of the drug resistance detection results of DY-210 and commonly used clinical antibacterial drugs.

[0032] Figure 5 Molecular docking mode diagram of DY-210 and the target PmtCD. Detailed implementation mode

[0033] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0034] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and do not have to be used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described here can be implemented in an order different from those illustrated or described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0036] 1. Synthesis of the compound

[0037] In the following experimental operations, all reactions involving air- or moisture-sensitive compounds were carried out under an argon atmosphere. Unless otherwise stated, all reagents and solvents were purchased from commercial suppliers without further purification, and anhydrous solvents were transferred using a syringe. Purification of the products was generally performed by silica gel (200 - 300 mesh) column chromatography. 1 1H NMR was measured on a Bruker ADVANCE II (400 MHz) using deuterated chloroform or deuterated dimethyl sulfoxide as the solvent and tetramethylsilane (TMS) as the internal standard.

[0038] Example 1

[0039] Synthesis of 1-(8-cyclohexyl-1,2,3a,4,5,6-hexahydro-3H-pyrazino[3,2,1-jk]carbazol-3-yl)-2-(4-methylpiperazin-1-yl)ethan-1-one (DY-210)

[0040] Dissolve 6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one (15.8 g, 60 mmol) in DMF, add NaH (4.8 g, 120 mmol), stir at room temperature for 30 min, then add chloroacetamide (11.2 g, 120 mmol), react at room temperature overnight, stop the reaction after TLC detection shows that the raw materials have basically disappeared, pour the reaction solution into ice water for quenching, extract with EA (3 × 100 ml), combine the organic phases, wash with a large amount of water, dry over anhydrous Na2SO4, concentrate to obtain the crude product, and purify by column chromatography to obtain the target product 2-(6-bromo-1-oxo-1,2,3,4-tetrahydro-9H-carbazol-9-yl)acetamide, which is a white solid, with a total of 13.8 g and a yield of 72%. 1 1H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 1.9 Hz, 1H), 7.51 (dd, J = 8.9, 1.9 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 5.13 (s, 2H), 3.00 (t, J = 6.1 Hz, 2H), 2.68 (dd, J = 7.2, 5.7 Hz, 2H), 2.25 (p, J = 6.3 Hz, 2H), 1.60 (s, 2H).

[0041] The above-obtained 2-(6-bromo-1-oxo-1,2,3,4-tetrahydro-9H-carbazol-9-yl)acetamide (13.8 g, 43 mmol) was added to anhydrous THF, and NaBH4 (6.5 g, 172 mmol) was slowly added under an ice bath. The mixture was stirred at room temperature for 4 h. After the reaction was completed as detected by TLC, it was quenched with water, and the aqueous phase was extracted with DCM (3 × 30 ml). The organic phases were combined, concentrated to obtain a crude product, and purified by column chromatography to obtain a white solid, which was 8-bromo-3a,4,5,6-tetrahydro-1H-pyrazino[3,2,1-jk]carbazol-2(3H)-one (10 g, yield 76%). 1 H NMR (400 MHz, DMSO-d6) δ 7.63 - 7.53 (m, 2H), 7.29 (t, J = 7.3 Hz, 2H), 7.22 (dd, J = 8.6, 2.0 Hz, 1H), 5.28 (d, J = 5.7 Hz, 1H), 4.92 - 4.68 (m, 3H), 2.70 (dt, J = 15.8, 4.4 Hz, 1H), 2.02 - 1.05 (m, 6H).

[0042] The above-obtained 8-bromo-3a,4,5,6-tetrahydro-1H-pyrazino[3,2,1-jk]carbazol-2(3H)-one (10 g, 32.7 mmol) was dissolved in anhydrous THF, and a THF solution of 2.5 M cyclohexylmagnesium bromide (78.5 ml, 196.2 mmol) and Pd(dppf)Cl2 (2.4 g, 3.27 mmol) were added. Argon was filled, and the mixture was heated and stirred at 80 °C overnight. It was quenched with saturated ammonium chloride, extracted with EA, the organic phases were combined and washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated, and then separated and purified by column chromatography to obtain a white solid, which was 8-cyclohexyl-2,3,3a,4,5,6-hexahydro-1H-pyrazino[3,2,1-jk]carbazole (5.2 g, yield 52%). 1 H NMR (400 MHz, DMSO-d6) δ 7.51 (s, 1H), 7.32 - 7.15 (m, 3H), 6.99 (d, J = 8.4 Hz, 1H), 5.23 (d, J = 5.3 Hz, 1H), 4.84 - 4.65 (m, 3H), 2.70 (d, J = 15.9 Hz, 1H), 1.99 - 1.66 (m, 9H), 1.37 (dtd, J = 51.2, 24.0, 11.8 Hz, 6H).

[0043] The obtained 8-cyclohexyl-2,3,3a,4,5,6-hexahydro-1H-pyrazino[3,2,1-jk]carbazole (5.2 g, 16.8 mmol) was dissolved in anhydrous THF, and 2.5 M LAH (4 ml, 100 mmol) was added under an ice bath. The mixture was heated under reflux at 60 °C for 10 h. After the reaction was detected by TLC to be complete, water, 10% NaOH solution, and water were added sequentially to quench the reaction. The mixture was filtered through diatomaceous earth, concentrated, and purified by column chromatography to obtain 8-cyclohexyl-2,3,3a,4,5,6-hexahydro-1H-pyrazino[3,2,1-jk]carbazole, a total of 3.8 g, with a yield of 78%. 1 HNMR (400 MHz, Chloroform-d) δ 7.55 (d, J = 7.8 Hz, 1H), 7.21 - 7.15 (m, 1H), 7.13 - 7.07 (m, 1H), 4.88 (dt, J = 10.8, 3.3 Hz, 1H), 4.41 - 4.03 (m, 3H), 3.30 - 2.46 (m, 11H), 2.23 - 1.13 (m, 8H).

[0044] The obtained 8-cyclohexyl-2,3,3a,4,5,6-hexahydro-1H-pyrazino[3,2,1-jk]carbazole (80 mg, 0.2 mmol), 4-methyl-1-piperazineacetic acid (32 mg, 0.2 mmol), and TBTU (60 mg, 0.21 mmol) were added to a round-bottom flask. After stirring evenly, Et3N (0.04 ml, 0.5 mmol) was added under an ice bath. After addition, the mixture was allowed to warm to room temperature naturally and stirred overnight. After the reaction was detected by TLC to be complete, saturated ammonium chloride was added to quench the reaction. The mixture was extracted with DCM (3 × 30 ml), and the organic phases were combined, concentrated, and purified by column chromatography to obtain the target product 1-(8-cyclohexyl-1,2,3a,4,5,6-hexahydro-3H-pyrazino[3,2,1-jk]carbazol-3-yl)-2-(4-methylpiperazin-1-yl)ethan-1-one, namely DY-210, as an off-white solid, a total of 30 mg, with a yield of 35%. 11H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.03 (dd, J = 8.4, 1.3 Hz, 1H), 5.14 (dd, J = 12.5, 5.7 Hz, 1H), 4.10 - 4.12 (m, 2H), 3.42 - 3.52 (m, 2H), 3.02 (s, 2H), 2.78 - 2.67 (m, 2H), 2.60 - 2.34 (m, 9H), 2.25 (s, 3H), 2.17 (ddd, J = 8.6, 7.2, 3.3 Hz, 1H), 1.96 - 1.81 (m, 7H), 1.54 - 1.35 (m, 4H), 1.32 - 1.25 (m, 1H).

[0045] Example 2

[0046] Synthesis of 1-(8-Cyclohexyl-1,2,3a,4,5,6-hexahydro-3H-pyrazino[3,2,1-jk]carbazol-3-yl)-2-(diethylamino)ethan-1-one (DY-217)

[0047] Weigh 8-cyclohexyl-2,3,3a,4,5,6-hexahydro-1H-pyrazino[3,2,1-jk]carbazole (100 mg, 0.34 mmol), ethylglycine (45 mg, 0.34 mmol), and TBTU (110 mg, 0.34 mmol) obtained in Example 1-1, add them to a round-bottom flask, add DCM under argon protection, slowly add Et3N (0.12 ml, 0.85 mmol) under ice bath, slowly raise the temperature to room temperature after addition and stir overnight, add saturated ammonium chloride to quench the reaction, extract with DCM, combine the organic phases, concentrate, and purify by column chromatography to obtain a yellow solid, which is 1-(8-cyclohexyl-1,2,3a,4,5,6-hexahydro-3H-pyrazino[3,2,1-jk]carbazol-3-yl)-2-(diethylamino)ethan-1-one, with a total of 38 mg and a yield of 28%. 11H NMR (400 MHz, Chloroform-d) δ 7.62 (dt, J = 13.1, 3.9 Hz, 1H), 7.35 - 7.30 (m, 1H), 7.10 - 7.03 (m, 1H), 4.95 (dt, J = 13.0, 3.4 Hz, 1H), 4.43 - 4.17 (m, 2H), 3.82 - 3.57 (m, 2H), 2.90 - 2.83 (m, 2H), 2.80 (s, 3H), 2.58 (tq, J = 11.6, 4.6, 3.3 Hz, 2H), 2.38 (qd, J = 7.1, 4.7 Hz, 4H), 2.12 (tdd, J = 11.0, 7.6, 4.8 Hz, 1H), 2.01 - 1.72 (m, 7H), 1.56 - 1.17 (m, 6H), 0.87 (td, J = 7.1, 4.1 Hz, 6H).

[0048] Example 3

[0049] Synthesis of (8-cyclohexyl-1,2,3a,4,5,6-hexahydro-3H-pyrazino[3,2,1-jk]carbazol-3-yl)(3-isopropyl-1H-pyrazol-5-yl)methanone (DY-218)

[0050] Weigh out 8-cyclohexyl-2,3,3a,4,5,6-hexahydro-1H-pyrazino[3,2,1-jk]carbazole (100 mg, 0.34 mmol) obtained in Example 1-1, 3-isopropyl-1H-pyrazole-5-carboxylic acid (52 mg, 0.34 mmol), and TBTU (110 mg, 0.34 mmol), add them to a round-bottom flask, add DCM under argon protection, slowly add Et3N (0.12 ml, 0.85 mmol) in an ice bath, and slowly raise the temperature to room temperature after addition and stir overnight. Quench the reaction with saturated ammonium chloride, extract with DCM, combine the organic phases, concentrate, and purify by column chromatography to obtain a yellowish-brown solid, namely (8-cyclohexyl-1,2,3a,4,5,6-hexahydro-3H-pyrazino[3,2,1-jk]carbazol-3-yl)(3-isopropyl-1H-pyrazol-5-yl)methanone, a total of 45 mg, with a yield of 31%. 11H NMR (400 MHz, Chloroform-d) δ 7.71 (t, J = 5.6 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.18 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.10 - 7.04 (m, 1H), 6.47 (d, J = 2.8 Hz, 1H), 4.94 (dt, J = 10.5, 3.3 Hz, 1H), 4.48 - 4.12 (m, 4H), 3.72 (ddq, J = 53.0, 12.4, 6.1 Hz, 3H), 2.95 - 2.42 (m, 5H), 2.10 - 1.71 (m, 8H), 1.18 (dd, J = 6.9, 2.1 Hz, 11H).

[0051] The preparation processes of other tetrahydrocarbazolopyrazine small molecule compounds refer to the above Examples 1 - 3, and the specific synthesis method conditions are summarized in Table 1 as follows:

[0052] Table 1

[0053]

[0054]

[0055]

[0056]

[0057] 2. Evaluation of antibacterial activity of compounds

[0058] 2.1 Determination of the minimum inhibitory concentration (MIC) of compounds

[0059] Dissolve the compounds of the present invention in DMSO, with the mother liquor concentration of 2 mg / ml, and store at -20 °C. Pipette 512 μl of the stock solution into 488 μl of M - H broth medium. After mixing, the highest concentration of the antibacterial drug is 2048 μg / ml. Pipette 100 μl of the highest - concentration drug into the first well of each row, and serially dilute it to the 10th well in a two - fold dilution manner, so that the gradient concentrations of the drug are 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2 μg / ml in turn. Pipette 100 μl of the diluted bacterial solution into the 96 - well plate in turn, and the final inoculated bacterial concentration is 5 × 10 5, place the 96-well plate on a micro oscillator and shake for 1 min to mix the bacterial suspension in each well, and incubate at 35 °C in a wet box for 16 - 20 h. Meanwhile, set drug-free growth control wells and sterile control wells for each group. The sterile control wells should remain clear throughout the experiment, indicating that the entire laboratory is under aseptic operation. The lowest drug concentration without visible growth compared with the growth characteristics of bacteria in the growth control wells is the MIC of the drug against the test bacteria.

[0060] The experimental results are shown in Table 2, indicating that the compound DY-210 of the present invention has good inhibitory effects on Staphylococcus aureus strains 29213, LAC, and Mu50. In particular, the compound DY-210 exhibits good antibacterial activity, and the MIC can reach 4 μg / ml. Therefore, DY-210 was selected for subsequent experiments to deeply evaluate its antibacterial activity.

[0061] Table 2

[0062]

[0063] 2.2 Determination of bacterial growth curve

[0064] Take 4 sterile conical flasks, labeled respectively as "growth control tube, drug administration group (2 μg / ml DY-210), drug administration group (4 μg / ml DY-210), drug administration group (8 μg / ml DY-210)", and add 6 ml of nutrient broth to each flask. Add a quantitative test drug to each conical flask, and add the same volume of deionized water to the growth control group. Add 60 μl of the test bacterial suspension with a concentration of 0.5 McFarland standard to each conical flask, and the final concentration of the bacterial suspension in each tube is about 10 6 CFU / ml. Immediately after adding the bacteria, vortex each conical flask evenly for 15 s. After serial dilution 1000-fold in a 10-fold ratio, take 0.1 ml of the bacterial suspension and inoculate it on M-H agar plates (two replicates for each), incubate overnight at 35 °C and then perform colony counting, and take the average value. After culturing at 37 °C for 2, 4, 6, 8, and 10 h respectively, vortex each conical flask evenly for 15 s, and perform serial dilution 10 0 ~10 7 times in a 10-fold ratio as above. Then take 0.1 ml of the bacterial suspension and inoculate it on M-H agar plates (three replicates for each), incubate overnight at 35 °C, and count the colonies on the plates with the number of colonies between 30 - 300, and take the average value. CFU = average colony number of the four plates × dilution factor, which is the number of viable bacteria per milliliter of the bacterial suspension in the original solution. Count the colonies at different time points and calculate their average values, and draw a bactericidal curve at different time points on logarithmic coordinate paper.

[0065] The experimental results are as Figure 1 shown. The results indicate that compared with the control group, 4 μg / ml of DY-210 has an impact on the three tested Staphylococcus aureus strains 29213 ( Figure 1In A), LAC( Figure 1 In B), Mu50( Figure 1 In C), the growth curves all showed obvious inhibitory activities, and 8 μg / ml DY-210 could basically completely inhibit the growth of bacteria.

[0066] 2.3 Bacterial electron microscopy experiment

[0067] 2.3.1 Scanning electron microscopy

[0068] Take the cryopreserved LAC bacteria, streak-inoculate them on M-H agar medium, and incubate them in an incubator (35 ± 2 °C) for 20 - 24 h. The next day, pick single colonies and inoculate them into 2 ml of nutrient broth, and shake the bacteria overnight at 180 rpm and 37 °C. The bacterial liquid in the logarithmic growth phase is adjusted to a concentration of 0.5 McFarland turbidity standard with M-H broth medium. Take 4 sterile test tubes and label them as "control, 30, 60, and 90 min" respectively. Add 2 ml of the bacterial liquid with 0.5 McFarland turbidity to each tube, and add antibacterial drugs to the latter 3 tubes so that their final concentrations are all 24 μg / ml. Add the same volume of deionized water to the control tube. Incubate and shake in a shaker at 120 rpm and 37 °C. After 30 min, take out the "30 min" tube and place it in a 4 °C refrigerator; after 60 min, take out the "60 min" tube and place it in a 4 °C refrigerator; after 90 min, take out the "90 min and control" tubes and place them in a 4 °C refrigerator. Take out the 4 tubes of bacterial liquid in the refrigerator, transfer them into 1.5 ml centrifuge tubes respectively, centrifuge at 2500 rpm for 5 min, and discard the supernatant. Wash the precipitate 2 times with 0.01 M PBS, centrifuge at 2500 rpm for 5 min, and discard the supernatant. Resuspend the precipitate with 100 μl of 0.01 M PBS, then add 500 μl of 3% glutaraldehyde for pre-fixation for more than 2 h. After fixation, rinse 3 times with 0.1 M phosphate buffer, and then fix with 1% osmium tetroxide fixing solution for 2 h. Dehydrate successively with 50%, 70%, 80%, 90%, and 95% acetone gradients for 15 min each, and dehydrate with amyl acetate for another 15 min. Freeze-dry the sample, use an ion sputtering instrument to form a metal film on the surface of the sample, and observe with a Hitachi S-3400N scanning electron microscope.

[0069] The results are as Figure 2 shown in A. The results showed that compared with the control group, under the action of 2 μg / ml DY-210, the surface of the bacteria shrank, the morphology was irregular, and gradually showed a swelling phenomenon. Under the action of 4 μg / ml DY-210, the test bacteria showed obvious swelling and rupture.

[0070] 2.3.2 Transmission electron microscopy

[0071] The cryopreserved LAC bacteria were inoculated on M-H agar medium and incubated in an incubator (35 ± 2°C) for 20 - 24 h. The next day, single colonies were picked and inoculated into 2 ml of nutrient broth, and the bacteria were shaken overnight at 180 rpm and 37°C. The bacterial suspension in the logarithmic growth phase was adjusted to a concentration of 0.5 McFarland standard with M-H broth medium. Two conical flasks were taken and labeled as "control group, drug administration group" respectively. 12 ml of the bacterial suspension with 0.5 McFarland turbidity was added to each conical flask. An antibacterial agent was added to the "drug administration group" to make its final concentration 24 μg / ml. The "control group" was added with the same volume of deionized water. The mixtures were shaken and cultured in a shaker at 120 rpm and 37°C for 90 min. The bacterial suspensions in the two conical flasks were respectively transferred into 1.5 ml centrifuge tubes and centrifuged at 2500 rpm for 5 min, and the supernatant was discarded. The precipitate was washed twice with 0.01 M PBS, and finally centrifuged at 2500 rpm, and the supernatant was discarded. 500 μl of 3% glutaraldehyde was added to the precipitate and pre-fixed overnight in a 4°C refrigerator. Then it was fixed with 1% osmium tetroxide fixing solution for 1 - 2 h. After fixation, it was rinsed 3 times with 0.1 M phosphoric acid buffer solution and stained with 1% uranyl acetate for 2 h. Dehydration was carried out successively with 50%, 70%, 80%, 90% and 100% acetone for 15 min each; first, it was soaked in a mixture of acetone and embedding medium (1:1) in an oven at 37°C for 30 min, then soaked in a mixture of acetone and embedding medium (1:4) in an oven at 37°C overnight, and finally soaked in pure embedding medium in an oven at 45°C for 2 h. The mixed embedding medium was first dropped into a rubber embedding plate, then the tissue block was transferred in and a bar code was placed, and finally the mixed embedding agent was filled. It was first placed in an oven at 45°C for polymerization for 3 h, and then placed in an oven at 65°C for polymerization for 48 h. Ultra-thin sections of conventional transmission electron microscope samples were made and stained with uranyl acetate and lead citrate, and observed with a JEM-2000EX transmission electron microscope.

[0072] The experimental results are as Figure 2 shown in B below. The results show that compared with the control group, under the action of 2 μg / ml DY-210, the bacterial cell membrane became damaged. Under the action of 4 μg / ml DY-210, the bacterial cell membrane ruptured, the internal differentiation was serious, and a large number of bacteria died, indicating that DY-210 has good antibacterial activity.

[0073] 2.4 In vivo antibacterial activity test of the compound

[0074] 2.4.1 Protective effect of DY-210 on sepsis in BALB / c mice caused by MRSA

[0075] Inoculate LAC into nutrient broth medium. After culturing at 37 °C for 16 - 18 h, dilute the LAC bacterial solution with 5% gastric mucin to enhance its virulence. Select healthy BALB / c mice at 8 - 10 weeks of age and weighing 18 - 22 g, with 16 mice in each group, half male and half female. Dilute the freshly cultured LAC 10-fold with 5% gastric mucin into 5 concentrations, and intraperitoneally inject and infect 4 mice with each concentration. Observe the death status of the animals to determine the bacterial quantity.

[0076] Experimental grouping and treatment: Randomly divide 18 mice into 3 groups, with 6 mice in each group. Intraperitoneally inject each mouse with LAC suspension (8.5×10 5 CFU / ml, 20 mg / kg). Different treatments are given at 1, 6, 20, and 28 h after infection: ① Control group: No treatment with any therapeutic drug; ② DY-210 treatment group: Intraperitoneally inject DY-210 (10 mg / kg / time); ③ Oxacillin treatment group: Intraperitoneally inject oxacillin (10 mg / kg / time). Continuously observe 6 BALB / c mice each time for 10 d and record the death situation.

[0077] The experimental results are as Figure 3 shown in A. Compared with the blank group and the oxacillin control group, treatment with 10 mg / kg DY-210 can significantly improve the survival rate of mice infected with LAC, indicating that DY-210 also has good antibacterial activity in vivo.

[0078] 2.4.2 Protective effect of DY-210 on tissues of BALB / c mice caused by LAC

[0079] Tissue section: After intercepting part of the liver, spleen, lung, and kidney and rinsing them with physiological saline, soak them in 40 g / L formalin fixative for 24 h. Embed the tissues in wax and then section them for HE staining.

[0080] Staining of mouse liver, spleen, lung, and kidney tissues: After the fixed tissues are dehydrated through a gradient of 70%, 80%, 90%, 95%, 95%, 100%, 100%, 100% alcohol, cleared with xylene, and infiltrated with wax to make wax blocks, section them at a thickness of 5 μm, stick the sections, and after baking, perform the following steps of dewaxing to water and staining:

[0081] Steps of dewaxing to water: ① Dewax with xylene I for 5 min; ② Dewax with xylene II for 5 min; ③ Dewax with xylene III for 5 min; ④ Wash with absolute ethanol for 1 min; ⑤ Wash with 95% ethanol I for 1 min; ⑥ Wash with 95% ethanol II for 1 min; ⑦ Wash with 80% ethanol for 1 min; ⑧ Rinse with tap water for 3 - 5 min.

[0082] Staining steps: ① Harris hematoxylin solution for 3 - 5 minutes; ② Rinse with tap water for 1 minute; ③ Differentiate with 75% hydrochloric acid ethanol solution for several seconds; ④ Rinse with running tap water for 5 minutes until the cell nuclei turn blue; ⑤ 0.5% eosin aqueous solution for 1 minute; ⑥ Dehydrate successively with 95% ethanol I and ethanol II for 1 minute each; ⑦ Dehydrate successively with absolute ethanol I and absolute ethanol II for 1 minute each; ⑧ Clear successively with xylene I and xylene II for 1 minute each; ⑨ Mount with neutral gum and observe tissue characteristics under a biological microscope.

[0083] The experimental results are as Figure 3 shown in B of [reference]. The results indicate that, compared with the model group, under the treatment of 10 mg / kg DY - 210 drug, the pathological damage of the lungs, liver, and kidneys of mice was significantly reversed and approached the normal level. It further demonstrated the good therapeutic effect of DY - 210 in vivo.

[0084] 2.5 Compound drug resistance experiment

[0085] The operation steps include: determining the MIC value of the compound against LAC, using the same method as in 1. Dilute the bacteria in the above 1 / 2MIC wells to a concentration of 5×10 5 CFU / ml, which is the bacteria for sub - culture. Determine the MIC value of the antibacterial drug against LAC again, using the same method as in 2.1. For 30 consecutive days, measure the MIC value of the antibacterial drug against LAC once a day, using the same method as above. Calculate the relative value of the MIC of the antibacterial drug against the bacteria cultured for 15 generations to the MIC of the bacteria cultured for the 1st generation.

[0086] The experimental results are as Figure 4 shown. Compared with the clinically commonly used antibacterial drugs oxacillin, ampicillin, and vancomycin, DY - 210 is not easily induced to produce bacterial drug resistance, showing its advantages different from existing antibiotics and good application prospects.

[0087] 2.6 Research on the mechanism of action of the compound

[0088] 2.6.1 Sensitization experiment of compound DY - 210 to natural antibacterial peptide LL - 37

[0089] Inoculate Staphylococcus aureus in Mueller - Hinton broth and culture overnight at 37°C. Adjust the bacterial liquid concentration to 0.5 McFarland turbidity (about 1×10 8 CFU / mL) with sterile normal saline. Dissolve the natural antibacterial peptide LL - 37 and the small - molecule compound DY - 210 separately with sterile normal saline to prepare stock solutions with appropriate concentrations. Perform serial two - fold dilutions of LL - 37 and DY - 210. In a 96 - well plate, add the serially diluted LL - 37 horizontally and the serially diluted DY - 210 vertically to form a checkerboard pattern. Add 100 μL of the adjusted bacterial liquid to each well to make the final bacterial liquid concentration 5×105 CFU / mL. Set up negative controls (containing only medium and bacterial solution), positive controls (containing only medium and LL-37), and blank controls (containing only medium). Incubate at 37 °C for 18 - 24 h, and observe and record the minimum inhibitory concentration (MIC) of each well.

[0090] The experimental results are shown in Table 3. In the presence of DY-210, the MIC of LL-37 against LAC changed from the original 256 μg / ml to 64 μg / ml, indicating that DY-210 has a relatively obvious sensitizing activity on the natural antimicrobial peptide LL-37, suggesting that DY-210 may play an antibacterial role by inhibiting the natural antimicrobial peptide efflux pump.

[0091] Table 3

[0092]

[0093] 2.6.2 Antibacterial activity test of compound DY-210 against Δpsm LAC strain

[0094] Inoculate the Δpsm LAC strain into Mueller-Hinton broth and incubate at 37 °C overnight. Adjust the concentration of the bacterial solution to 0.5 McFarland turbidity (about 1×10 8 CFU / mL) with sterile normal saline. Dissolve the small molecule compound DY-210 with sterile normal saline to prepare a stock solution with an appropriate concentration. Perform serial two-fold dilutions in a 96-well plate to set up wells with different drug concentrations. In the 96-well plate, add 100 μL of the adjusted bacterial solution to each well to make the final bacterial solution concentration 5×10 5 CFU / mL. Set up negative controls (containing only medium and bacterial solution) and blank controls (containing only medium). After incubating at 37 °C for 18 - 24 h, read the lowest drug concentration without bacterial growth, which is the minimum inhibitory concentration (MIC) of the strain against the drug.

[0095] The experimental results (see Table 4) show that in the Δpsm LAC strain, the antibacterial activity of DY-210 decreased, and the MIC decreased by 1-fold, suggesting that DY-210 may play an antibacterial activity by targeting the related pathway of the secretion of the key toxin PSM of Staphylococcus aureus, initially verifying the mechanism of action of the compound designed in the present invention.

[0096] Table 4

[0097]

[0098] 2.6.3 Molecular docking of compound DY-210 with the target PmtCD

[0099] First, obtain the crystal structure of PmtCD (PDB-6XJI) from the PDB database, and use AutoDock Tools to remove water molecules, add hydrogen atoms, and optimize the protonation state. After constructing the 3D structure of compound DY-210 with ChemDraw, perform geometric optimization and energy minimization using Gaussian. Before docking, use the AutoDock Vina software to define the active pocket and set the docking box. Adjust the parameters when running the docking (such as Vina's exhaustiveness = 32), and evaluate the conformational changes of DY-210 through semi-flexible docking. Sort the docking results according to the binding free energy (ΔG), and use PyMOL to analyze the key interactions (hydrogen bonds, hydrophobic interactions, π-π stacking, etc.), and focus on observing the binding mode of DY-210 to the catalytic residues of PmtCD. Finally, verify the stability of the complex through molecular dynamics simulation (GROMACS / AMBER), and verify the binding affinity by combining MM-PBSA calculations.

[0100] The docking results (see Figure 5 ) show that the tetrahydrocarbazolopiperazine tetracyclic structure core in compound DY-210 occupies the cavity of the target protein, forming good spatial complementarity. Among them, the carbonyl oxygen atom in DY-210 forms a hydrogen bond interaction with the protein residue Gly38 as a hydrogen bond acceptor; while the nitrogen atom on the piperidine ring can, on the one hand, form a salt bridge with the protein residue Asp144; on the other hand, it can act as a hydrogen bond donor and form a hydrogen bond interaction with the protein residue Glu145; in addition, the hydrophobic cyclohexyl group and part of the aromatic ring system in DY-210 can form van der Waals forces with adjacent protein residues.

[0101] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution according to the technical idea proposed by the present invention fall within the protection scope of the claims of the present invention.

Claims

1. Use of a class of tetrahydrocarbazole-piperazine small molecule compounds or salts thereof in the preparation of antibacterial drugs or antibacterial drug sensitizers, characterized in that, The structure of the tetrahydrocarbazolopyrazine small molecule compound is shown in the following formula I: Wherein, n is 0, 1, 2 or 3; R1, R2, R3 and R4 are any one of hydrogen, halogen, lower haloalkane, lower alkane, lower cycloalkane, hydroxyl, lower hydroxyalkane, lower alkoxy, phenyl, substituted phenyl, amino, lower alkylamino, lower haloalkylamino, lower cycloalkylamino, lower alkynylamino, nitro, lower nitroalkyl, cyano, lower cyanoalkyl, amide, lower cycloalkylamide and lower amidealkyl; R5 is hydrogen or an organic ketone compound; The *-marked position is a chiral carbon atom.

2. The application according to claim 1, characterized in that, The organic ketone compounds include 1-((1H-1,2,4-triazol-5-yl)thio)propan-2-one, 1-morpholinopropan-2-one, (1-adamantan-1-yl)ethan-1-one, 1-(benzod[d]oxazol-2-ylthio)propan-2-one, 1-(4,6-dimethylpyrimidin-2-yl)thio)propan-2-one, 1-((5-amino-1,3,4-thiadiazol-2-yl)thio)propan-2-one, 1-(4-fluorophenyl)propan-2-one, 4-acetyl-N,N-dipropylbenzenesulfonamide, 1-(4-phenylpiperazin-1-yl)propan-2-one, 1-(4-nitrophenyl)ethan-1-one, 1-(4-methylpiperazin-1-yl)propan-2-one, 7-(methylsulfonyl)-3,4-dihydroquinolin-2(1H)-one, N-phenylethanethioamide, N-(4-butoxyphenyl)ethanethioamide, 5-(methylsulfonyl)benzo[d]thiazol-2(3H)-one, pent-1-yne, 1-(piperidin-1-yl)butan-2-ol, but-1-yne, 4-(4-fluorophenyl)butan-2-one, 1-(4-(2-methoxyphenyl)piperazin-1-yl)propan-2-one, 1-(4-(3-chlorophenyl)piperazin-1-yl)propan-2-one, acetophenone, 1-(2-phenoxyphenyl)ethan-1-one, 1-(phenylthio)propan-2-one, 1-(dimethylamino)propan-2-one, furan-3-yl(4-(2-hydroxybutyl)piperazin-1-yl)methanone, 4-(3,4-dimethoxyphenyl)-2-methyl-4,5-dihydrothiazole, 1-(piperidin-1-yl)propan-2-one, 1-(diethylamino)propan-2-one and 1-(3-isopropyl-1H-pyrazol-5-yl)ethan-1-one.

3. The application according to claim 1, characterized in that The lower cycloalkane is a ring containing 3 to 7 carbons; the lower substituent means that the corresponding aliphatic hydrocarbon group is straight-chain or branched-chain, saturated, and contains 1 to 4 carbon atoms.

4. The application according to claim 1, characterized in that, The tetrahydrocarbazolopyrazine small molecule compound is:

5. The application according to claim 1, wherein The tetrahydrocarbazolopyrazine small molecule compound reacts with an acid to form a salt, and the acid includes hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, acetic acid, tartaric acid, salicylic acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid or succinic acid.

6. The application according to claim 1, wherein The drug is an antibacterial drug against Gram-positive bacteria.

7. The application according to claim 6, wherein The Gram-positive bacteria are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

8. The application according to claim 7, wherein Tetrahydrocarbazolopyrazine small molecule compounds and their salts exert antibacterial effects by acting on the target PmtCD.

9. The application according to claim 7, wherein Tetrahydrocarbazolopyrazine small molecule compounds and their salts exert antibacterial effects by inhibiting the natural antibacterial peptide efflux pump.

10. The application according to claim 7, characterized in that Tetrahydrocarbazolopyrazine small molecule compounds and their salts exert dual antibacterial activities by inhibiting the secretion of key toxin PSM and the efflux of natural antibacterial peptides.

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